Alpha (2->8) polysialic acid (PSA) forms the capsular polysaccharide (CPS) of group B meningococci (GBM), Escherichia coli K1 (ECK1) and a part of E. coli K92 CPS and is associated with pathogenesis and protection in GBM and ECK1 infections. PSA is a poorly immunogenic, conformationally complex T-independent antigen. To evaluate the complexity of immunodeterminants expressed on this CPS and to identify epitope(s) with biologic functions, two panels of murine monoclonal antibodies (mAbs) were generated and characterized by immunizing BALB/c mice with GBPS-OMP (fusion A) or OAc+K1-OMP (fusion B) conjugate vaccine. Fusion A yielded 23 mAbs representative of IgM class and all subclasses of IgG and showed 6 different microspecificities as examined by their differential binding to native and chemically modified PSA attached to a solid phase by various methods; fusion B yielded 11 mAbs with microheterogenous specificities. Confirmatory competitive inhibition ELISA revealed that the mAbs recognized a mosaic of immunodeterminants on OAc+K1PS and GBPS. Two mAbs induced by OAc+K1-OMP conjugate were directed to O-acetyl epitope and the rest reacted with OAc+K1 and only with GBPS attached to solid phase in certain forms. MAbs generated by the GBPS-OMP conjugate mostly recognized an epitope shared by both GBPS and OAc+K1PS; one also recognized an epitope on ECK92. IgG1 mAbs and those with O-acetyl specificity were non-bactericidal to GBM in presence of rabbit complement, whereas other mAbs were bactericidal. No correlation could be established between the microspecificity of mAbs and their in vitro bactericidal activity. Conjugation and complexation of PSA with proteins appear to improve the accessibility of certain epitopes for mAb binding. Thus the methods by which antigen was coated on solid phase had a great impact on the binding specificity and intensity of mAbs. GBPS antibodies from group B meningococcal patients, carriers and vaccinees also showed similar microheterogeneity in epitope binding. Immunogenicity tests done in mice revealed that both GBPS-OMP and OAc+K1-OMP conjugates were immunogenic and produced both IgG and IgM capsular antibodies. The adjuvant MPL significantly enhanced the capsular antibody response. Antibodies induced by OAc+K1-OMP conjugate were mostly O-acetyl epitope-specific. Temperature dependence in antigen binding and various other properties of conjugate-induced polyclonal GBPS antibodies are being evaluated.